EFFECT OF PH AND ENZYME LOADING ON THE PROTEIN

CONCENTRATION DURING COLLOCALIA FUCIPHAGA HYDROLYSIS

AHMAD AFIFI BIN IBRAHIM

A thesis submitted in fulfilment

of the requirements for the award of the Degree of

Bachelor of Chemical Engineering (Biotechnology)

Faculty Of Chemical & Natural Resources Engineering

Universiti Malaysia Pahang

FEBRUARY 2013

vi

ABSTRACT

The purpose of this study is to investigate the effect of pH and enzyme loading

on the concentration of protein during enzymatic hydrolysis of Collocalia Fuciphaga. C.

Fuciphaga is refers to the bird nest from one swiftlet species that is used as the sample

known to be highly nutritious. Bovine Serum Albumin (BSA), a standard protein

solution was used as the benchmark for the reference of the protein concentration. A

research had been proposed in this study to optimize the production of protein from

C.Fuciphaga. The effect of changing of the parameters values during the enzymatic

hydrolysis was studied and the higher protein concentrations produced were highlighted.

The standard protein curve of Bovine Serum Albumin (BSA) is prepared first with

several dilutions for protein standard curve. The results from two significant parameters

in the experiment was taken and analyzed. The selection of parameters values was taken

from the enzymes’ optimum condition where the pH value of Alcalase 2.4L is from pH

7 until pH 10 whiles the temperature is fixed at 50oC. Enzyme loading was varies from

1.5% to 3.0% (v/w). The sample was analyzed using UV-Vis Spectrophotometer to

determine the concentration of protein in each sample. The optimum condition is found

to be at extraction condition of pH 8.5 and enzyme concentration of 3.0% (v/w).

Characterization of the sample had been done and validated the protein composition on

the extracted sample.

vii

ABSTRACT

Tujuan kajian ini adalah untuk mengkaji kesan pH dan kepekatan enzim pada

kepekatan protein semasa hidrolisis enzim Collocalia Fuciphaga. C. Fuciphaga adalah

merujuk kepada sarang burung dari satu spesies burung walit yang digunakan sebagai

sampel kerana ia dikenali untuk menjadi sangat berkhasiat. Serum Albumin Bovine

(BSA), larutan protein piawai telah digunakan sebagai penanda aras untuk rujukan

kepekatan protein. Satu penyelidikan telah dicadangkan dalam kajian ini untuk

mengoptimumkan pengeluaran protein dari C.Fuciphaga. Kesan perubahan nilai

parameter semasa hidrolisis enzim telah dikaji dan kepekatan protein yang lebih tinggi

yang dihasilkan telah diketengahkan. lengkung protein standard Bovine Serum Albumin

(BSA) disediakan terlebih dahulu dengan pencairan beberapa sebagai lengkung protein

piawai. Keputusan dari dua parameter signifikan dalam eksperimen akan diambil dan

dianalisis. Pemilihan nilai parameter telah diambil dari keadaan optimum enzim di mana

nilai pH Alcalase 2.4L adalah dari pH 7 sehingga pH 10 manakala suhu ditetapkan pada

50oC. Keadaan optimum didapati berada pada keadaan pengekstrakan pH 8.5 dan

kepekatan enzim sebanyak 3.0% (v / w). Sampel ini kemudiannya akan dianalisis

menggunakan Spectrophotometer UV-Vis untuk menentukan kepekatan protein dalam

setiap sampel. Pencirian sampel telah dilakukan dan mengesahkan komposisi protein

pada sampel yang diekstrak.

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TABLE OF CONTENT

CHAPTER TITLE PAGE

TITLE PAGE i

SUPERVISOR DECLARATION ii

STUDENT DECLARATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

LIST OF FIGURES xii

LIST OF TABLES xiv

LIST OF ABBREVIATION xv

LIST OF SYMBOL xvi

1 INTRODUCTION

1.1 Background of Proposed Study 1

1.2 Problem Statement 3

1.3 Objectives 4

1.4 Scope of Proposed Study 4

1.5 Significance of Proposed Study 5

2 LITERATURE REVIEW

2.1 Sources of Protein 7

2.1.1 Plant as a Sources of Protein 8

2.1.2 Animal Source of Protein 9

2.1.3 Amino Acid in Animal 10

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2.1.4 Types of Amino Acids 11

2.2 Application of Amino Acid 13

2.2.1 Advantages of Amino Acid 14

2.2.2 Structural View of Amino Acid 14

2.3 Protein from Animal 15

2.3.1 Edible Bird’s Nest 16

2.3.1.1 Nutritional Value of EBN 17

2.3.2 Reason Selection of Animal Protein 18

2.3.3 Problem Related on Protein Consumption 19

2.4 Process Protein Production 20

2.4.1 Biological Method : Enzymatic Hydrolysis 22

2.5 Parameters 23

2.5.1 Effect of pH on the protein concentration 23

in Enzymatic Hydrolysis.

2.5.2 Effect of Temperature on the protein concentration 23

in Enzymatic Hydrolysis.

2.5.3 Effect of Enzyme loading on the protein

concentration in Enzymatic Hydrolysis. 24

2.6 Analysis Methods 24

2.6.1 DNS method 24

2.6.2 Lowry Method Analysis 25

2.6.3 Fourier Transform Infrared Spectroscopy 25

3 METHODOLOGY

3.1 Introduction 26

3.1.1 Stage I : Pre-Treatment Stage 27

3.1.2 Stage II : Enzymatic Hydrolysis Stage 29

3.1.2.1 Extraction Process 30

3.1.2.2 Enzyme Addition 31

3.1.2.3 One Factor at a Time (OFAT) Method 32

3.1.2.4 Incubation of Sample Solution 33

x

3.1.2.5 The Separation of the Mixture 33

3.1.3 Step III : Sample Analysis 34

3.2 Materials 35

3.2.1 Collocalia fuciphaga 35

3.2.2 Protease Enzyme 35

3.3 Methods 36

3.3.1 Prepare the Standard Protein Curve 36

3.3.2 Preparation of Buffer Solutions 36

3.3.3 Protein Extraction 37

3.3.4 Solid-Liquid Separation 39

3.3.5 Determination Protein Concentration in Sample 40

3.3.6 Characterization of the Sample Using 41

Fourier Transform Infrared Spectroscopy (FTIR)

3.3.7 Flow Diagram on Overall Process 43

4 RESULTS AND DISCUSSION

4.1 Introduction 44

4.2 Protein Standard Curve 45

4.3 Effects of pH on The Protein Content 46

4.4 Effects of Temperature to the Hydrolysis Process 50

4.5 Effect of Enzyme Loading on The Protein Yield 51

4.6 Chemical Characterization Of The Processed EBN 55

By FTIR

5 CONCLUSION AND RECOMMENDATION

5.1 Conclusion 58

5.2 Recommendation 59

xi

REFERENCES 60-63

APPENDIX

Appendix A 64

Appendix B 66

Appendix C 67

Appendix D 68

xii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

Figure 2-1 Example of different sources of protein 8

Figure 2-2 Twenty amino acid overview diagram 13

Figure 2-3 Amino acid molecular unit 15

Figure 2-4 Collocalia fuciphaga nest 16

Figure 2-5 FTIR Sample Analysis Process 17

Figure 3-1 Overall Stages of Research 27

Figure 3-2 Untreated raw material 28

(Collocalia fuciphaga bird nest)

Figure 3-3 Pre-treatment of raw material 28

Figure 3-4 Removal of feathers and plumage 28

Figure 3-5 Sample filtrations 29

Figure 3-6 Overview process of sample pre-treatment 29

Figure 3-7 Overview process of enzymatic hydrolysis 30

Figure 3-8 Extraction process of sample 31

Figure 3-9 Extraction process 32

Figure 3-10 Water bath BS-21 33

Figure 3-11 The flow of the research methodology 39

Figure 3-12 Sorvall RC5C centrifuge 40

Figure 3-13 UV-Vis Spectrophotometers 41

Figure 3-14 Sample characterization using FTIR 42

Figure 3-15 Overall process on research methodology 43

Figure 4-1 Optical Density (OD) versus BSA concentration 45

Figure 4-2 Protein concentration versus pH of the sample 47

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solution in first trial.

Figure 4-3 Protein concentration versus pH of the sample 47

solution in second trial.

Figure 4-4 Protein concentration of versus pH of the sample 48

solution in third trial.

Figure 4-5 Average value on protein concentration yield on 48

different pH

Figure 4-6 FTIR analysis results on different pH solution. 49

(a) pH 7 (b) pH 8 (c) pH 9 (d) pH 10

Figure 4-7 Concentration of protein versus enzyme loading 51

(%) of the sample solutions in first trial.

Figure 4-8 Protein concentration versus enzyme loading (%) 52

of the sample solutions in second trial.

Figure 4-9 Protein concentration versus enzyme loading (%) 52

of the sample solutions in third trial.

Figure 4-10 Average protein concentration versus enzyme 53

loading (%) of the sample solutions for three trials.

Figure 4-11 FTIR analysis on different enzyme loading of 55

1.5% (v/w) and 3.0 % (v/w)

Figure 4-12 Comparison of analysis using FTIR from 56

processed EBN sample from (a) pH 7 to (b) pH 10

Figure 4-13 Available groups of compounds detected by on 57

FTIR spectrum analysis on protein yield from

processed EBN sample.

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LIST OF TABLES

TABLE TITLE PAGE

Table 2-1 Twenty types of amino acids with classification 12

Table 2-2 Mean (SEM) concentration of protein, minerals 18

and sialic acid in processed EBN samples

Table 2-3 Protein quality ranking 19

Table 2-4 Pre-treatment methods for enzymatic hydrolysis 21

Table 3-1 Dilution from the BSA solution (1.0 g/L) for 36

the standard curve

Table 3-2 Data for the one factor at a time (OFAT) 38

Table 4-1 Optical Density of Different Concentration of 45

Bovine Serum Albumin

Table 4-2 Concentration of protein from samples from 47

extraction on different pH solutions

Table 4-3 Concentration of protein yield from sample from 51

extraction with different enzyme loading

Table A-1 Preparation of 0.1 M Sodium Acetate Buffer 64

Table A-2 Preparation of 0.1 M Potassium Phosphate 65

Buffer

Table A-3 Preparation of 0.1 M Glycine-NaOH Buffer 65

Table B-1 IR Absorptions for Representative Functional 67

Groups

Table C-1 Amino acid distribution (mg/g) of edible bird's 68

nest extract.

Table D.1 Reagents for Modified Lowry’s Method 69

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LIST OF ABBREVIATIONS

EBN Edible Bird Nest

FTIR Fourier Transform Infrared Spectroscopy

OFAT One Factor at a Time

DNS Dinitrosalicylic acid

BSA Bovine Serum Albumin

AU Anson Unit

NaOH Sodium Hyroxide

OD Optical Density

rpm Round per Minute

SEM Scanning Electron Microscopy

xvi

LIST OF SYMBOLS

oC - Degree Celsius

% - Percent

g - Gram

L - Liter

rpm - Rotation Per Minutes

M - Molarity

ml - Milliliter

v - Vibration wavelength

cm-1

- Reciprocal Centimeter

mg - Milligram

Abs - Absorbance

ml - Millilitre

min - Minute

hr - Hour

CHAPTER 1

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Proteins are essential food components because they are a source of amino acids

needed for growth and maintenance and provide functional properties to foods.

Commercially available protein foods are obtained from a range of animal and plant

sources and are used as functional ingredients (Martina. 2002). Due to the increasing

costs and limited supplies of animal proteins and since vegetable protein is the most

abundant source of protein on the Earth, a number of vegetable proteins such as alfalfa

leaf, cottonseed, winged bean, peanut and soya have been investigated for possible

incorporation into formulated foods.

Protein molecules tend to unfold and even become fully denatured under

unfavoured conditions, such as a high temperature, an acidified condition, a high

pressure or even excessive shear. Denatured protein molecules will aggregate and/or

2

crosslink to form larger clusters and, at high concentrations, will form a three-

dimensional solid-like network (or a gel). Therefore, proteins are regarded as one of the

main classes of building blocks used in many semi-solid foods for conferring

mechanical properties (Linqiang et. al., 2008).

For this particular research, the raw material that will be used is Collocalia

Fuciphaga. C. fuciphaga species is popularly known as the White Nest or House Nest

swiftlet. C. fuciphaga measures about 12 cm in its entire length and weighs about 15 to

18 gm. In recent years, hormone-like substances such as mitogen and avian epithelial

growth factor have been found in C. fuciphaga (Jie et al., 2009).

Edible bird’s nest (EBN) is the nest of the swift and is constructed with salivary

glue, which is a cementing substance, and may incorporate other materials such as

vegetation or feathers. Although EBN mainly contains carbohydrates, amino acids, and

mineral salts, the abundant ingredient is composing of glycoproteins (Wu , Wu et al.,

2010). Due to its nutritious and medical properties, EBN has been deemed a precious

food tonic in Chinese community ever since the Tang (907 AD) and Sung (960–1279

AD) dynasties. Despite the long history of using EBN for medicinal purpose, there have

only been a limited number of scientific reports on the health benefits of EBN.

3

1.2 PROBLEM STATEMENT

The problem of the research is to find out the most appropriate condition that can

optimize the protein yield via the enzymatic hydrolysis process. The important part is to

search for the protein sources that can be produced in a great amount so that the

nutrients from the food can be obtained by all people who in need. Hence the nutritional

food is widespread produced all around the world to get all of the healthy life with all of

those nutritional benefits. However, the protein extracted from marine life, sometimes

not suitable to consumers with heart diseases and blood pressure.

With the researches that had been conducted nowadays, protein from various

sources; from plant or from other animal is being looked out. Besides, the most optimum

production of the nutritious food is being focused nowadays to produce an optimum

condition for the extraction of the protein. The research regarding the optimum condition

of the protein concentration of Collocalia fuciphaga has not been done before.

1.3 OBJECTIVES

The main objective of this research is to characterize and optimize protein

extraction yield from C.fuciphaga using enzymatic hydrolysis method. In this research

also have a few specific objectives. The specific objectives are:

4

To determine the influence of pH on extraction protein yield.

To determine the influence of enzyme loading on extraction protein yield.

To characterize the sample by using Fourier Transform Infrared

Spectroscopy (FTIR).

1.4 SCOPES OF THE STUDY

Below are the scopes of the study for the research that will be conducted;

This research will be focusing on the characterization of C.fuciphagamainly

using the Ultra-violet Visible spectrophotometer.

Study the effect of three parameters, the pH of the solution from pH 7-10,

and the enzyme loading ranging from 1.5% to 3.0

The optimal condition of pH, and enzyme loading during the enzymatic

hydrolysis will be determined by using Lowry Method One Factor at a Time

(OFAT).

5

1.5 SIGNIFICANCE OF THE PROPOSED STUDY

Nowadays the application of protease is often an attractive means for obtaining

better functional properties of food proteins, without impairing their nutritional value. In

order to increase and to improve the protein quality produced from the sources of

protein, the extracted protein need to undergo the hydrolysis process with the addition of

enzyme with several modifications made (Ng et al., 2012). The process of protein

extraction using enzymatic hydrolysis method is proved to produce hydrolysates with

well-defined peptide profiles (Palupi et al., 2010).

Hence, the study of the extraction protein from Collocalia Fuciphaga with the

optimum condition will be conducted so that the nutrients on the protein in the bird nest

will be preserve and produce higher protein yield. Besides people that doubtful with the

contents of the nest especially the Muslim and also Vegetarian users will leave the doubt

and take the benefits of the bird nest. As the sample taken is not from the marine life or

any animal sources, so it does not causes allergic to the consumer.

Hence it is really suitable for all type of people who need it.

CHAPTER 2

LITERATURE REVIEW

2.1 SOURCES OF PROTEIN

Protein is essential for the development of the human life as they are really

needed for the growth and maintenance of the body. They are sources of amino acids

that responsible for many functional properties that influence to foods. The basic

structures of protein are composed of small units. The units are consists of small units

which are the amino acids units that are also called the building blocks of protein.

Protein is an essential nutrient for human many living things. Protein is contained in

every part of your body, the skin, muscles, hair, blood, body organs, eyes, even

fingernails and bone. Protein is the most plentiful substance in your body after water and

no life can survive without protein (Lauritzen, 1992). There are various sources of

protein that can be taken nowadays from a two different type of protein sources.

7

The sources can be classified as the animal protein and the plant protein. Protein

does exist for both animal and plant sources. Different type of animal and plant sources

have different amount of protein in it. The determination of protein in various sources

had been discovered and continuously in the research to know the protein concentration

and the quality of the protein. Hence nowadays, a wide range of protein from animal and

plant are being commercialized and are available to be used today to be used as useful

ingredient for general and specific uses (Hoffman & Falvo, 2004). Protein is however

not a primary source of energy but it can be used as energy where the protein have to be

metabolized into the much more simplest form into the amino acid from in order to

generate energy. (Hoffman & Falvo, 2004)

Figure 2-1 Example of different Sources of Protein

2.1.1 Plant as a Sources of Protein

The source of protein from plant is being nowadays being the alternatives for the

consumption rather than the protein from animal. The protein on plants can be obtained

8

from a lot of sources. Whole grains and cereals are another source of protein. There are a

lot of sources of protein that can be obtained nowadays. Proteins from plants contribute

over 65 percent on the worldwide basis on the per capita supply of protein (Young &

Pellen, 1994) Oats, maize, rice and wheat are examples of protein sources which the

concentration of protein is greater than 7 percent (Young & Pellen, 1994). For the

vegetarian sources of protein the sources include the legumes, nuts, seeds and fruits.

Some of the vegetarian foods with higher concentration of protein include soybeans,

lentils, white beans, pigeon peas, almonds, sunflower seeds, cowpeas, walnuts and

pumpkin seeds.

Determination of protein in plant had been made in determining the quality and

the digestibility of the protein. The plant consider as incomplete protein because of the

protein is not having the all the amino acid that should be consumed in human body.

According to Hoffman & Falvo (2004), the protein form plant said to be having less

protein from animal protein and are generally lacking single or two amino acids.

Vegetable protein is the alternative choices for those who are really want to have a result

in a reduction in the intake of the saturated fat and cholesterol.

2.1.2 Animal Source of Protein

Proteins from animal sources is the highest quality food sources that been

consume on today’s life. The protein that are taken from protein is has the quality higher

than the protein from the plant sources. The protein from animal can be consumed from

9

a lot of sources such as meat, egg, fish, poultry and milk. These protein sources of food

have been consume by almost all of the human population around the world for the

consumption of the protein in their everyday diet. Based on the statement made by

(Hoffman & Falvo, 2004), the protein from animal has the highest quality rating due to

the protein in the animal has all the required characteristics as the protein needed. The

protein is said to be in the completeness for the animal protein. Also for the benefits

taken from total protein consumption, a lot of elderly people had also been benefited

from consuming animal sources of protein. Meat consisting diets had been resulted in

greater gains in lean body mass compared to subjects on a lactoovovegetarian diet

(Campbell et al., 1999).

The animal protein is said to be having a higher contents of protein than the

protein in the vegetable plant protein. A high animal protein diets in the daily intake had

been shown to give a much greater net protein synthesis than a high vegetable protein

diet (Pannemans et al., 1998). This statement supports the information that protein is

much better in the protein content rather than the plant protein. Hence the protein

content synthesis from the plant is less compare to the content of protein in the animal.

2.1.3 Amino Acid in Animal

Amino acid commonly known to be 20 different types of amino acids those are

different from each other. Each different protein is composed of various amino acids put

together in varying order with almost limitless combinations. Most proteins are large

molecules that may contain several hundred amino acids arranged in branches and

10

chains (Lauritzen, 1992). The protein sources can be divided into two major classes that

are plant sources and animal sources protein. The protein in animal is a complete source

of amino acid where all the amino acids that are essential to our body can be taken from

the animal sources. This shows that the protein in the plant generally lack one or more

amino acids in it. People who want to take benefits from the plant in term of taking the

protein from it have to take several type of vegetables or plant to take all the required

amino acids in their everyday diet (Hoffman & Falvo, 2004).

For the 20 amino acids that had been identified, they are all needed for human

growth and metabolism. However Hoffman and Flavo (2004) stated that in the total of

20 types of amino acids, twelve of these amino acids or eleven in children are

nonessential amino acid. The nonessential amino acid meaning that our body can

produce the amino acid on our own body without consuming in the everyday diet. The

synthesis of the amino acid can be synthesis by the human body by its own without

having to be taken from other sources. For the other remaining amino acids, they cannot

be synthesized in the body and are described as essential amino acid meaning that they

have to be consumed in our diets. Without any of these amino acids, problems will

occurs where it will compromise the ability of tissue to grow, be repaired or be

maintained (Hoffman & Falvo, 2004).

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2.1.4 Types of Amino Acids

There are several types of amino acid that are being the general classification of

the amino acids. There are classified based on the different functional group attached to

the amino acid with different location with one to another. The carboxylic acid group

and amine group attached at the on the same location to the first, or alpha, carbon atom

next to the –COOH group (Clark, 2011). The amino acid types can be classified based

on the general statement of the requirement on dietary scope of some amino acids.

However due to the different amount of the requirement of the amino acid, the

classification is hard to be made.

Table 2-1 Twenty Types of Amino Acids with Classification (Volpi et al., 2013)

Essential Nonessential

Histidine Alanine

Isoleucine Arginine

Leucine Aspartate

Lysine Asparagine

Methionine Glutamic acid

Phenylalanine Glutamine

Threonine Glycine

Valine Serine

Tyrosine

12

Figure 2-2 Twenty amino acid overview diagram (www.wikipedia.com)

2.2 APPLICATION OF AMINO ACID

The amino acid had been used in many industries especially the food industry.

The food industries had been benefited a lot from the technology regarding the amino